Manual positioning of small objects for microscopy is time-consuming. Finding an object, placing it centrally in the field of view and focusing on the features of interest accounts for a significant part of the workload associated with clinical microscopy. A particular case of interest is microscopy of oocytes and embryos as part of a standard IVF treatment where handling of the embryo outside the safe incubator environment with defined temperature, atmosphere and pH is a significant stress factor that must be minimized. Automated systems are known, which are able to re-position objects efficiently by seeking out predefined X-Y-Z coordinates where the object of interest is deemed to be positioned. However, the initial task of obtaining the correct coordinates for a given object is very time-consuming. If object movement and image acquisition is automated, it usually implies acquiring consecutive images in a grid pattern and stitching these together in a collage to enable search algorithms or the human operator to find the object of interest. Alternatively it involves the assistance of an operator to locate the object manually and record the coordinates.
While it is generally possible to position larger objects in predefined spots where they remain stationary, this becomes increasingly difficult with objects of microscopic sizes in a liquid media. An example of particular interest is early human embryos, which are only about ⅛ mm in diameter (approx. 120 μm) with a density slightly higher than their growth medium. Positioning embryos accurately in media droplets is difficult, and handling easily disturbs their position.
The exact positioning of a microscopic object is of general interest in many applications. However, when using Embryo Transfer (ET) techniques, such as IVF (In Vitro Fertilization) and related techniques, this involves in vitro culturing of the developing embryo for a period of days before re-implantation of selected embryos. Even with the ideal growth conditions, selection criteria are needed as a tool to choose the most viable embryos for re-implantation. The viability of an embryo is an important parameter in order to determine the embryos suitability for transfer. At a practical level, precise positioning for microscopy is a prerequisite for assessing the viability of the embryo following manipulation. In practice, embryo evaluation is limited to a more or less subjective grading based on morphological criteria. The respiration rate of the embryo may prove a good candidate for an objective viability indicator. Therefore, a need exists for a fast, simple and non-disturbing method, system and device for enabling morphological evaluation and/or possibly measurement of individual embryo respiration rates as a measure for the embryo viability.
WO2001/02539 describes a well within a well (WOW) approach to provide individual cell cultures in microscopy slides, an advantage thereof as stated is providing a small volume well within a larger volume well for enabling a better culturing environment for each individual embryo.
However, manual and/or automatic handling of the microscopy slide comprising said WOW have proven to be time consuming and difficult, because it is practise has proven to be difficult to insert an embryo or other cell culture in the smaller volume well. Further if one was to provide more than one WOW upon a slide there may be a risk of contamination from one WOW to another WOW, which may be detrimental to cell growth. One problem in particular being that if one cell culture experiences lysis or other developmental problems, a contamination or spread of cell lysate from one well to another may propagate such problem.
Another problem with known microscopy slides, e.g. comprising at least one WOW, for culturing purposes is how to maintain a stable environment for developing said living cell cultures, i.e. keeping a relatively stable predetermined temperature, a stable gas composition, a sufficient amount of culturing media, the required humidity, and reduce the risk of contamination from the surroundings, and other environmental influences.
Further, even though a more accurate positioning of individual objects may be provided using WOW devices due to the fact that a relatively determined coordinate set in both the x, y and z direction may be accomplished within said slide; in view of the manipulating problems stated above, the use of a WOW device may reduce the handling time, but not significantly, nor does it protect against mutual and external contamination.
When applied to embryo viability assessment, the miscoscopy method, device and system according to the system should be providing the following key elements as outlined by Overström 1996 (see In vitro assessment of embryo viability. Theriogenology 45:3-16 1996): 1) The ability to make simultaneous objective measurements of multiple individual embryos, 2) The sensitivity and resolution to measure individual embryos/oocytes, 3) Rapid evaluation i.e. ˜30 min or less, 4) Viability test must be non-perturbating and ideally non-invasive, 5) Technically simple and user friendly, 6) Reduced cost when acquiring and handling.
On the above background it is one purpose of the present invention to provide a method, a device and a system of the initially mentioned type, which reduces the above mentioned disadvantages of the know methods, devices and systems.